Human CTLA-8 and uses of CTLA-8-related proteins

ABSTRACT

Polynucleotides encoding human CTLA-8 and related proteins are disclosed. Human CTLA-8 proteins and methods for their production are also disclosed. Methods of treatment using human CTLA-8 proteins, rat CTLA-8 proteins and herpesvirus herpes CTLA-8 proteins are also provided.

This application is a divisional of application Ser. No. 08/685,239,filed Jul. 18, 1996, now abandoned, which is a continuation-in-part ofapplication of Ser. No 08/504,032 filed Jul. 19, 1995, now converted toProvisional application No. 60/035347 filed Jul. 19, 1995, and acontinuation-in-part of application Ser. No. 08/514,014, filed Aug. 11,1995, now U.S. Pat. No. 5,707,829.

FIELD OF THE INVENTION

The present invention relates to human CTLA-8 proteins, nucleic acidsencoding such proteins, methods of treatment using such proteins. Theinvention also relates to the use of rat CTLA-8 proteins and herpesvirusSaimiri 0RF13 proteins in methods of treatment.

BACKGROUND OF THE INVENTION

Cytokines are secreted proteins which act on specific hematopoietictarget cells to cause a differentiation event or on other target cellsto induce a particular physiological response, such as secretion ofproteins characteristic of inflammation. Cytokines, also variously knownas lymphokines, hematopoietins, interleukins, colony stimulatingfactors, and the like, can be important therapeutic agents, especiallyfor diseases or conditions in which a specific cell population isdepleted. For example, erythropoietin, G-CSF, and GM-CSF, have allbecome important for treatment of anemia and leukopenia, respectively.Other cytokines such as interleukin-3, interleukin-6, interleukin-11 andinterleukin-12 show promise in treatment of conditions such asthrombocytopenia and modulation of immune response.

For these reasons a significant research effort has been expended insearching for novel cytokines and cloning the DNAs which encode them. Inthe past, novel cytokines were identified by assaying a particular cellsuch as a bone marrow cell, for a measurable response, such asproliferation. The search for novel cytokines has thus been limited bythe assays available, and if a novel cytokine has an activity which isunmeasurable by a known assay, the cytokine remains undetectable. In anewer approach, cDNAs encoding cytokines have been detected using thepolymerase chain reaction (PCR) and oligonucleotide primers havinghomology to shared motifs of known cytokines or their receptors. The PCRapproach is also limited by the necessity for knowledge of previouslycloned cytokines in the same protein family. Cytokines have also beencloned using subtractive hybridization to construct and screen cDNAlibraries, or they can potentially be cloned using PCR followed by gelelectrophoresis to detect differentially expressed genes. Thesubtractive hybridization methods are based on the assumption thatcytokine mRNAs are those that are differentially expressed, and thesemethods do not require any prior knowledge of the sequence of interest.However, many cytokines may be encoded by mRNAs which are notdifferentially expressed, and thus are undetectable using these methods.

It would be desirable to develop new methods for identifying novelcytokines and other secreted factors and to isolate polynucleotidesencoding them.

SUMMARY OF THE INVENTION

In developing the present invention, methods were employed whichselectively identify polynucleotides which encode secreted proteins. Onesuch polynucleotide was isolated which encodes "human CTLA-8." Inaccordance with the present invention, polynucleotides encoding humanCILA-8 and active fragments thereof are disclosed. "CTLA-8" is usedthroughout the present specification to refer to both proteins andpolynucleotides encoding those proteins and to refer to proteins andpolynucleotides from all mammalian species.

In certain embodiments, the present invention provides an isolatedpolynucleotide comprising a nucleotide sequence selected from the groupconsisting of:

(a) the nucleotide sequence of SEQ ID NO: 1 from nucleotide 146 tonucleotide 544;

(b) a nucleotide sequence capable of hybridizing to a nucleic acidsequence specified in (a);

(c) a nucleotide sequence varying from the sequence of the nucleotidesequence specified in (a) as a result of degeneracy of the genetic code;and

(d) an allelic variant of the nucleotide sequence specified in (a).Preferably, the polynucleotide of the invention encodes a protein havingCTLA-8 activity. In other embodiments the polynucleotide is operablylinked to an expression control sequence. In other preferredembodiments, the polynucleotide is contained in a vector suitable for invivo expression in a mammalian subject. Polynucleotides comprising thenucleotide sequence of SEQ ID NO: 1 from nucleotide 55 to nucleotide544, the nucleotide sequence of SEQ ID NO: 1 from nucleotide 139 tonucleotide 544 or the nucleotide sequence of SEQ ID NO: 1 fromnucleotide 86 to nucleotide 544 are particularly preferred.

Host cells transformed with the polynucleotides of the invention arealso provided, including mammalian cells.

Processes are also provided for producing a human CTLA-8 protein, saidprocesses comprising:

(a) growing a culture of the host cell of the invention in a suitableculture medium; and

(b) purifying the human CTLA-8 protein from the culture.

Isolated human CTLA-8 protein is also provided which comprising an aminoacid sequence selected from the group consisting of:

(a) the amino acid sequence of SEQ ID NO:2;

(b) the amino acid sequence of SEQ ID NO:2 from amino acids 11 to 163;

(c) the amino acid sequence of SEQ ID NO:2 from amino acids 29 to 163;

(d) the amino acid sequence of SEQ ID NO:2 from amino acids 31 to 163;and

(e) fragments of (a), (b), (c) or (d) having CTLA-8 activity.

Proteins comprising the amino acid sequence of SEQ ID NO:2 andcomprising the sequence from amino acids 29 to 163, from amino acid 31to 163, or from amino acids 11 to 163 of SEQ ID NO:2 are particularlypreferred. Preferably, the protein has CTLA-8 activity. Pharmaceuticalscomposition comprising a human CTLA-8 protein of the invention and apharmaceutically acceptable carrier are also provided.

Compositions are also disclosed which comprise an antibody whichspecifically reacts with a human CTLA-8 protein of the invention.

Methods of treating a mammalian subject are also provided which compriseadministering a therapeutically effective amount of a pharmaceuticalcomposition comprising a human CTLA-8 protein.

Rat CTLA-8 and active (i.e., having CTLA-8 activity) fragments thereofmay also be used in such methods of treatment. Preferably the ratprotein is administered as a composition comprising a pharmaceuticallyacceptable carrier and a protein comprising an amino acid sequenceselected from the group consisting of:

(a) the amino acid sequence of SEQ ID NO:4;

(b) the amino acid sequence of SEQ ID NO:4 from amino acids 18 to 150;and

(c) fragments of (a) or (b) having CTLA-8 activity.

Herpesvirus Saimiri ORF13, referred to herein as "herpes CTLA-8", andactive (i.e., having CTLA-8 activity) fragments thereof and activefragments thereof may also be used in such methods of treatment.Preferably the herpes CTLA-8 protein is administered as a compositioncomprising a pharmaceutically acceptable carrier and a proteincomprising an amino acid sequence selected from the group consisting of:

(a) the amino acid sequence of SEQ ID NO:6;

(b) the amino acid sequence of SEQ ID NO:6 from amino acids 19 to 151;and

(c) fragments of (a) or (b) having CTLA-8 activity.

The invention also provides a method of treating a mammalian subjectcomprising administering a therapeutically effective amount of acomposition comprising a pharmaceutically acceptable carrier and IL-17or an active fragment thereof.

In methods of treatment provided by the present invention, preferablythe subject is treated to produce an effect selected from the groupconsisting of inhibition of angiogenesis, inhibition of growth orproliferation of vascular endothelial cells, inhibition of tumor growth,inhibition of angiogenesis-dependent tissue growth, proliferation ofmyeloid cells or progenitors, proliferation of erythroid cells orprogenitors, proliferation of lymphoid cells or progenitors, inductionof IFNγ production, induction of L-3 production and induction of GM-CSFproduction.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a comparison of homologous regions of the amino acid sequencesof human CTLA-8 (indicated as "B 18₋₋ F1"), rat CTLA-8 (indicated as"Musctla8") and herpes CTLA-8 (indicated as "Hsvie₋₋ 2").

FIG. 2 depicts autoradiographs demonstrating expression of human CTLA-8in COS cells.

FIG. 3 presents data relating to the ability of human CTLA-8 to inhibitangiogenesis.

FIGS. 4 and 5 present data relating to the ability of human CTLA-8 toproduce or induce hematopoietic activity.

FIGS. 6 and 7 present data demonstrating the ability of human CILA-8 toinduce production of L-6 and L-8.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The inventors of the present application have identified and provided apolynucleotide encoding a human CTLA-8 protein. SEQ ID NO:1 provides thenucleotide sequence of a cDNA encoding the human CTLA-8 protein. SEQ IDNO:2 provides the amino acid sequence of the human CTLA-8 protein.Alternatively, the initiating methionine may be at amino acid 11 of SEQID NO:2. On the basis of amino terminal sequencing, the mature proteinsequence is believed to begin at amino acid 31 of SEQ ID NO:2 (encodedby the sequence beginning with nucleotide 146 of SEQ ID NO: 1).

The region from amino acid 29 to amino acid 163 of human CTLA-8 (SEQ IDNO:2) shows marked homology to portions of rat CTLA-8 (amino acids 18 to150 of SEQ ID NO:4) and herpesvirus Saimiri ORF13 ("herpes CTLA-8")(amino acids 19 to 151 of SEQ ID NO:6). A cDNA sequence encoding ratCTLA-8 is listed at SEQ ID NO:3 and its corresponding amino acidsequence is reported at SEQ ID NO:4. A cDNA sequence encoding herpesCTLA-8 is listed at SEQ ID NO:5 and its corresponding amino acidsequence is reported at SEQ ID NO:6. Homology between rat CTLA-8 andherpes CTLA-8 was reported by Rouvier et al., J. Immunol. 1993, 150,5445-5456.

Applicants had previously incorrectly identified the rat sequences ofSEQ ID NO:3 and SEQ ID NO:4 as applying to murine CTLA-8. Applicants'human CTLA-8 (B 18) does also show homology to the true murine CTLA-8sequence.

Golstein et al. (WO95/18826; Fossiez et al., Microbial Evasion andSubversion of Immunity 544:3222 (Abstract)) have also reported a speciesthey initially identified as "human CTLA-8." However, examination of thesequence of the Golstein et al. species and the human CTLA-8 (B18)sequence of the present invention readily reveals that they are twodifferent proteins, although they are homologous with each other andwith the rat CTLA-8 and herpes CTLA-8 identified herein. The Golstein etal. species has now been renamed as interleukin-17 (IL-17). Because ofthe homology between applicants' human CTLA-8 (B118) and IL-17, theseproteins are expected to share some activities.

It has also been preliminarily determined that human CTLA-8 (B18) formshomodimers when expressed. As a result, human CTLA-8 proteins maypossess activity in either monomeric or dimeric forms. Human CTLA-8proteins can also be produced as heterodimers with rat and herpes CTLA-8proteins and with human IL-17. These heterodimers are also expected tohave activities of the proteins of which they are comprised.

Forms of human CTLA-8 protein of less than full length are encompassedwithin the present invention and may be produced by expressing acorresponding fragment of the polynucleotide encoding the human CTLA-8protein (SEQ ID NO: 1). These corresponding polynucleotide fragments arealso part of the present invention. Modified polynucleotides asdescribed above may be made by standard molecular biology techniques,including site-directed mutagenesis methods which are known in the artor by the polymerase chain reaction using appropriate oligonucleotideprimers.

For the purposes of the present invention, a protein has "CTLA-8activity" if it either (1) displays biological activity in afactor-dependent cell proliferation assay (preferably an assay in whichfull-length the corresponding species full-length CTLA-8 is active)(including without limitation those assays described below), or (2)induces expression or secretion of γ-IFN, or (3) displayschemoattractant or chemotactic activity in a chemoattraction orchemotaxis assay (preferably as assay in which full-length thecorresponding species full-length CTLA-8 is active) or (4) inducesexpression or secretion of IL-3 or GM-CSF.

Human CTLA-8 protein or fragments thereof having CTLA-8 activity may befused to carrier molecules such as immunoglobulins. For example, humanCTLA-8 protein may be fused through "linker" sequences to the Fc portionof an immunoglobulin.

The invention also encompasses allelic variations of the nucleotidesequence as set forth in SEQ ID NO: 1, that is, naturally-occurringalternative forms of the isolated polynucleotide of SEQ ID NO:1 whichalso encode human CTLA-8 or CTLA-8 proteins having CTLA-8 activity. Alsoincluded in the invention are isolated polynucleotides which hybridizeto the nucleotide sequence set forth in SEQ ID NO: 1 under highlystringent (0.2xSSC at 65° C.), stringent (e.g. 4×SSC at 65 C or 50%formamide and 4xSSC at 42° C.), or relaxed (4×SSC at 50° C. or 30-40%formamide and 4xSSC at 42° C.) conditions. Isolated polynucleotideswhich encode human CTLA-8 protein but which differ from the nucleotidesequence set forth in SEQ ID NO:1 by virtue of the degeneracy of thegenetic code are also encompassed by the present invention. Variationsin the nucleotide sequence as set forth in SEQ ID NO:1 which are causedby point mutations or by induced modifications which enhance CTLA-8activity, half-life or production level are also included in theinvention.

The isolated polynucleotides of the invention may be operably linked toan expression control sequence such as the pMT2 or pED expressionvectors disclosed in Kaufman et al., Nucleic Acids Res. 19, 4485-4490(1991), in order to produce the CTLA-8 protein recombinantly. Manysuitable expression control sequences are known in the art. Generalmethods of expressing recombinant proteins are also known and areexemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990). Asdefined herein "operably linked" means enzymatically or chemicallyligated to form a covalent bond between the isolated polynucleotide ofthe invention and the expression control sequence, in such a way thatthe CTLA-8 protein is expressed by a host cell which has beentransformed (transfected) with the ligated polynucleotide/expressioncontrol sequence.

A number of types of cells may act as suitable host cells for expressionof the human CTLA-8 protein. Any cell type capable of expressingfunctional human CTLA-8 protein may be used. Suitable mammalian hostcells include, for example, monkey COS cells, Chinese Hamster Ovary(CHO) cells, human kidney 293 cells, human epidermal A431 cells, humanColo205 cells, 3T3 cells, CV-1 cells, other transformed primate celllines, normal diploid cells, cell strains derived from in vitro cultureof primary tissue, primary explants, HeLa cells, mouse L cells, BHK,HL-60, U937, HaK, Rat2, BaF3, 32D, FDCP-1, PC12 or C2C12 cells.

The human CTLA-8 protein may also be produced by operably linking theisolated polynucleotide of the invention to suitable control sequencesin one or more insect expression vectors, and employing an insectexpression system Materials and methods for baculovirus/insect cellexpression systems are commercially available in kit form from, e.g.,Invitrogen, San Diego, Calif., U.S.A. (the MaxBac® kit), and suchmethods are well known in the art, as described in Summers and Smith,Texas Agricultural Experiment Station Bulletin No. 1555 (1987),incorporated herein by reference. Soluble forms of the human CTLA-8protein may also be produced in insect cells using appropriate isolatedpolynucleotides as described above.

Alternatively, the human CTLA-8 protein may be produced in lowereukaryotes such as yeast or in prokaryotes such as bacteria. Suitableyeast strains include Saccharomyces cerevisiae, Schizosaccharomycespombe, Kluyveromyces strains, Candida, or any yeast strain capable ofexpressing heterologous proteins. Suitable bacterial strains includeEscherichia coli, Bacillus subtilis, Salmonella typhimurium, or anybacterial strain capable of expressing heterologous proteins.

The human CTLA-8 protein of the invention may also be expressed as aproduct of transgenic animals, e.g., as a component of the milk oftransgenic cows, goats, pigs, or sheep which are characterized bysomatic or germ cells containing a polynucleotide sequence encoding thehuman CTLA-8 protein.

The human CTLA-8 protein of the invention may be prepared by growing aculture of transformed host cells under culture conditions necessary toexpress the desired protein. The resulting expressed protein may then bepurified from the culture medium or cell extracts. Soluble forms of thehuman CTLA-8 protein of the invention can be purified from conditionedmedia. Membrane-bound forms of human CTLA-8 protein of the invention canbe purified by preparing a total membrane fraction from the expressingcell and extracting the membranes with a non-ionic detergent such asTriton X-100.

The human CTLA-8 protein can be purified using methods known to thoseskilled in the art. For example, the human CTLA-8 protein of theinvention can be concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. Following the concentration step, the concentratecan be applied to a purification matrix such as a gel filtration mediumAlternatively, an anion exchange resin can be employed, for example, amatrix or substrate having pendant diethylaminoethyl (DEAE) groups. Thematrices can be acrylamide, agarose, dextran, cellulose or other typescommonly employed in protein purification. Alternatively, a cationexchange step can be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Sulfopropyl groups are preferred (e.g., S-Sepharose® columns).The purification of the human CTLA-8 protein from culture supernatantmay also include one or more column steps over such affinity resins asconcanavalin A-agarose, heparin-toyopearl® or Cibacrom blue 3GASepharose®; or by hydrophobic interaction chromatography using suchresins as phenyl ether, butyl ether, or propyl ether; or byimmunoaffinity chromatography. Finally, one or more reverse-phase highperformance liquid chromatography (RP-HPLC) steps employing hydrophobicRP-HPLC media, e.g., silica gel having pendant methyl or other aliphaticgroups, can be employed to further purify the human CTLA-8 protein. Someor all of the foregoing purification steps, in various combinations orwith other known methods, can also be employed to provide asubstantially purified isolated recombinant protein.

Preferably, the human CTLA-8 protein is purified so that it issubstantially free of other mammalian proteins.

It is believed that human CTLA-8, active fragments and variants thereof,and CTLA-8 related proteins (such as, for example, rat CTLA-8 and herpesCTLA-8) (collectively "CTLA-8 proteins") possess or induce cytokineactivities. Human CTLA-8 expression correlated with γ-IFN expression ininduced primary cells and can induce the expression of IL-3 and/orGM-CSF, which expression can in turn produce effects associated with theinduced cytokine. Therefore, human CTLA-8 and CTLA-8 related proteinsmay have an effect on proliferation or function of myeloid cells,erythroid cells, lymphoid cells and their progenitors. Human CTLA-8proteins may also play a role in formation of platelets or theirprogenitors.

A protein of the present invention may exhibit cytokine, cellproliferation (either inducing or inhibiting) or cell differentiation(either inducing or inhibiting) activity or may induce production ofother cytokines in certain cell populations. Many protein factorsdiscovered to date, including all known cytokines, have exhibitedactivity in one or more factor dependent cell proliferation assays, andhence the assays serve as a convenient confirmation of cytokineactivity. The activity of a protein of the present invention isevidenced by any one of a number of routine factor dependent cellproliferation assays for cell lines including, without limitation, 32D,DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8, RB5, DA1,123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Assays for T-cell or thymocyte proliferation include without limitationthose described in: Current Protocols in Immunology, Ed by J. E.Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W Strober,Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, InVitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7,Immnologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500,1986; Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolliet al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., J.Immunol. 149:3778-3783, 1992; Bowman et al., J. Immunol. 152: 1756-1761,1994.

Assays for cytokine production and/or proliferation of spleen cells,lymph node cells or thymocytes include, without limitation, thosedescribed in: Polyclonal T cell stimulation, Kruisbeek, A. M. andShevach, E. M. In Current Protocols in Immunology. J. E. e.a. Coliganeds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; andMeasurement of mouse and human Interferon γ, Schreiber, R. D. In CurrentProtocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8,John Wiley and Sons, Toronto. 1994.

Assays for proliferation and differentiation of hematopoietic andlymphopoietic cells include, without limitation, those described in:Measurement of Human and Murine Interleukin 2 and Interleukin 4,Bottomly, K., Davis, L. S. and Lipsky, P. E. In Current Protocols inImmunology. J. E. e.a Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wileyand Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211,1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc.Natl. Acad. Sci. U.S.A. 80:2931-2938, 1983; Measurement of mouse andhuman interleukin 6 - Nordan, R. In Current Protocols in Immunology. J.E. e.a Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto.1991; Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986;Measurement of human Interleukin 11 -Bennett, F., Giannotti, J., Clark,S. C. and Turner, K. J. In Current Protocols in Immunology. J. E. e.aColigan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991;Measurement of mouse and human Interleukin 9 - Ciarletta, A., Giannotti,J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology.J. E. e.a Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto.1991.

Assays for T-cell clone responses to antigens (which will identify,among others, proteins that affect APC-T cell interactions as well asdirect T-cell effects by measuring proliferation and cytokineproduction) include, without limitation, those described in: CurrentProtocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.Margulies, E. M. Shevach, W Strober Pub. Greene Publishing Associatesand Wiley-Interscience (Chapter 3, In Vitro assays for Mouse LymphocyteFunction; Chapter 6, Cytokines and their cellular receptors; Chapter 7,Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad.Sci. USA 77:6091-6095, 1980; Weinbergeret al., Eur. J. Immun.11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takaiet al., J. Immunol. 140:508-512, 1988.

A protein of the present invention may also exhibit immune stimulatingor immune suppressing activity, including without limitation theactivities for which assays are described herein. A protein may beuseful in the treatment of various immune deficiencies and disorders(including severe combined immunodeficiency (SCID)), e.g., in regulating(up or down) growth and proliferation of T and/or B lymphocytes, as wellas effecting the cytolytic activity of NK cells and other cellpopulations. These immune deficiencies may be genetic or be caused byviral (e.g., HIV) as well as bacterial or fungal infections, or mayresult from autoirmune disorders. More specifically, infectious diseasescauses by viral, bacterial, fungal or other infection may be treatableusing a protein of the present invention, including infections by HIV,hepatitis viruses, herpes viruses, mycobacteria, leshmania, malaria andvarious fungal infections such as candida. Of course, in this regard, aprotein of the present invention may also be useful where a boost to theimmune system generally would be indicated, i.e., in the treatment ofcancer.

Autoimmune disorders which may be treated using a protein of the presentinvention include, for example, multiple sclerosis, systemic lupuserythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation,Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependentdiabetes mellitis, myasthenia gravis, graft-versus-host disease andautoimmune inflammatory eye disease. Such a protein of the presentinvention may also to be useful in the treatment of allergic reactionsand conditions, such as asthma or other respiratory problems. Otherconditions, in which immune suppression is desired (including, forexample, asthma and related respriatory conditions), may also betreatable using a protein of the present invention.

A protein of the present invention may also suppress chronic or acuteinflammation, such as, for example, that associated with infection (suchas septic shock or systemic inflammatory response syndrome (SIRS)),inflammatory bowel disease, Crohn's disease or resulting from overproduction of cytokines such as TNF or IL-1 (such as the effectdemonstrated by IL-11).

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Suitable assays for thymocyte or splenocyte cytotoxicity include,without limitation, those described in: Current Protocols in Immunology,Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, WStrober, Pub. Greene Publishing Associates and Wiley-Interscience(Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19;Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl.Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol.128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985;Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA78:2488-2492, 1981; Herrmann et al, J. Immunol. 128:1968-1974, 1982;Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol.137:3494-3500, 1986; Bowmanet al., J. Virology 61:1992-1998; Takai etal., J. Immunol. 140:508-512, 1988; Bertagnolli et al., CellularImmunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-3092,1994.

Assays for T-cell-dependent immunoglobulin responses and isotypeswitching (which will identify, among others, proteins that modulateT-cell dependent antibody responses and that affect Th1/Th2 profiles)include, without limitation, those described in: Maliszewski, J.Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitroantibody production, Mond, J. J. and Brunswick, M. In Current Protocolsin Immunology. J. E. e.a. Coligan eds. Vol 1 pp.3.8.1-3.8.16, John Wileyand Sons, Toronto. 1994.

Mixed lymphocyte reaction (MLR) assays (which will identify, amongothers, proteins that generate predominantly Thl and CTL responses)include, without limitation, those described in: Current Protocols inImmunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M.Shevach, W Strober, Pub. Greene Publishing Associates andWiley-Interscience (Chapter 3, In Vitro assays for Mouse LymphocyteFunction 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai etal., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.

Dendritic cell-dependent assays (which will identify, among others,proteins expressed by denritic cells that activate naive T-cells)include, without limitation, those described in: Guery et al., J.Immunol. 134:536-544, 1995; Inaba et al., Journal of ExperimenalMedicine 173:549-559, 1991; Macatonia et al., Journal of Immunology154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine182:255-260, 1995; Nair et al., Journal of Virology 67:4062-4069, 1993;Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal ofExperimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal ofClinical Investigation 94:797-807, 1994; and Inaba et al., Journal ofExperimental Medicine 172:631-640, 1990.

Assays for lymphocyte survival/apoptosis (which will identify, amongothers, proteins that prevent apoptosis after superantigen induction andproteins that regulate lymphocyte homeostasis) include, withoutlimitation, those described in: Darzynkiewicz et al., Cytometry13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca etal., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243,1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai etal., Cytometry 14:891-897, 1993; Gorczyca et al., International Journalof Oncology 1:639-648, 1992.

Assays for proteins that influence early steps of T-cell commitment anddevelopment include, without limitation, those described in: Antica etal., Blood 84:111-117, 1994; Fine et al., Cellular Immunology155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al.,Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.

A protein of the present invention may be useful in regulation ofhematopoiesis and, consequently, in the treatment of myeloid or lymphoidcell deficiencies. Even marginal biological activity in support ofcolony forming cells or of factor-dependent cell lines indicatesinvolvement in regulating hematopoiesis, e.g. in supporting the growthand proliferation of erythroid progenitor cells alone or in combinationwith other cytokines, thereby indicating utility, for example, intreating various anemias or for use in conjunction withirradiation/chemotherapy to stimulate the production of erythroidprecursors and/or erythroid cells; in supporting the growth andproliferation of myeloid cells such as granulocytes andmonocytes/macrophages (i.e., traditional CSF activity) useful, forexample, in conjunction with chemotherapy to prevent or treat consequentmyelo-suppression; in supporting the growth and proliferation ofmegakaryocytes and consequently of platelets thereby allowing preventionor treatment of various platelet disorders such as thrombocytopenia, andgenerally for use in place of or complimentarily to platelettransfusions; and/or in supporting the growth and proliferation ofhematopoietic stem cells which are capable of maturing to any and all ofthe above-mentioned hematopoietic cells and therefore find therapeuticutility in various stem cell disorders (such as those usually treatedwith transplantation, including, without limitation, aplastic anemia andparoxysmal nocturnal hemoglobinuria), as well as in repopulating thestem cell compartment post irradiation/chemotherapy, either in-vivo orex-vivo (i.e. in conjunction with bone marrow transplantation) as normalcells or genetically manipulated for gene therapy.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Suitable assays for proliferation and differentiation of varioushematopoietic lines are cited above.

Assays for embryonic stem cell differentiation (which will identify,among others, proteins that influence embyronic differentationhematopoiesis) include, without limitation, those described in:Johansson et al. Cellular Biology 15:141-51, 1995; Keller et al.,Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al.,Blood 81:2903-2915, 1993.

Assays for stem cell survival and differentiation (which will identify,among others, proteins that regulate lympho-hematopoiesis) include,without limitation, those described in: Methylcellulose colony formingassays, Freshney, M. G. In Culture of Hematopoietic Cells. R. I.Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, N.Y.1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992;Primitive hematopoietic colony forming cells with high proliferativepotential, McNiece, I. K. and Briddell, R. A. In Culture ofHematopoietic Cells. R. L Freshney, et aL eds. Vol pp. 23-39,Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., ExperimentalHematology 22:353-359, 1994; Cobblestone area forming cell assay,Ploemacher, R. E. In Culture ofHematopoietic Cells. R. I. Freshney, etal. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, N.Y. 1994; Long termbone marrow cultures in the presence of stromal cells, Spooncer, E.,Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R. I.Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, N.Y.1994; Long term culture initating cell assay, Sutherland, H. J. InCulture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp.139-162, Wiley-Liss, Inc., New York, N.Y. 1994.

CTLA-8 proteins are useful in the treatment of various immunedeficiencies and disorders (including SCID), e.g., in regulating (up ordown) growth, proliferation and/or activity of T and/or B lymphocytes,as well as the cytolytic activity of NK cells. These immune deficienciesmay be caused by viral (e.g., HIV) as well as bacterial infections, ormay result from autoimmune disorders. More specifically, infectiousdiseases caused by viral , bacterial, fungal or other infection may betreatable using CTLA-8 proteins, including infections by HIV, hepatitis,influenza, CMV, herpes, mycobacterium, leishmaniasis, malaria andvarious fungal infections (such as candida). Of course, in this regard,the CTLA-8 proteins may also be useful where a boost to the immunesystem generally would be indicated, i.e., in the treatment of cancer oras an adjuvant to vaccines. Autoimmune disorders which may be treatedusing factors of the present invention include, for example, multiplesclerosis, systemic lupus erythematosus, rheumatoid arthritis,autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmunethyroiditis, insulin dependent diabetes melitis and autoimmuneinflammatory eye disease. The CTLA-8 proteins are also expected to beuseful in the treatment of allergic reactions and conditions.

CTLA-8 proteins are also expected to have chemotactic activity A proteinor peptide has "chemotactic activity," as used herein, if it canstimulate, directly or indirectly, the directed orientation or movementof cells, including myeloid and lymphoid cells. Preferably, the proteinor peptide has the ability to directly stimulate directed movement ofcells (particularly T-cells). Whether a particular protein or peptidehas chemotactic activity for cells can be readily determined byemploying such protein or peptide in any known assay for cellchemotaxis.

CTLA-8 proteins also inhibit growth and proliferation of vascularendothelial cells. As a result, human CTLA-8 proteins are effective ininhibiting angiogenesis (i.e., vascular formation). This activity willalso be useful in the treatment of tumors and other conditions in whichangiogenesis in involved. Inhibition of angiogenesis by human CTLA-8proteins will also result in inhibition or prevention of the conditionto which normal angiogenesis would contribute.

Isolated CTLA-8 proteins, purified from cells or recombinantly produced,may be used as a pharmaceutical composition when combined with apharmaceutically acceptable carrier. Such a composition may contain, inaddition to CTLA-8 protein and carrier, diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials well known inthe art. The term "pharmaceutically acceptable" means a non-toxicmaterial that does not interfere with the effectiveness of thebiological activity of the active ingredient(s). The characteristics ofthe carrier will depend on the route of administration. Thepharmaceutical composition of the invention may also contain cytokines,lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, IL-1,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,IL-15,G-CSF, γ-IFN, stem cell factor, and erythropoietin. Thepharmaceutical composition may contain thrombolytic or anti-thromboticfactors such as plasminogen activator and Factor VIII. Thepharmaceutical composition may further contain other anti-inflammatoryagents. Such additional factors and/or agents may be included in thepharmaceutical composition to produce a synergistic effect with CTLA-8protein, or to minimize side effects caused by the CTLA-8 protein.Conversely, CTLA-8 protein may be included in formulations of theparticular cytokine, lymphokine, other hematopoietic factor,thrombolytic or anti-thrombotic factor, or anti-inflammatory agent tominimize side effects of the cytokine, lymphokine, other hematopoieticfactor, thrombolytic or anti-thrombotic factor, or anti-inflammatoryagent.

The pharmaceutical composition of the invention may be in the form of aliposome in which CTLA-8 protein is combined, in addition to otherpharmaceutically acceptable carriers, with amphipathic agents such aslipids which exist in aggregated form as micelles, insoluble monolayers,liquid crystals, or lamellar layers which in aqueous solution. Suitablelipids for liposomal formulation include, without limitation,monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids,saponin, bile acids, and the like. Preparation of such liposomalformulations is within the level of skill in the art, as disclosed, forexample, in U.S. Pat. No. 4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat.No. 4,837,028; and U.S. Pat. No. 4,737,323, all of which areincorporated herein by reference.

As used herein, the term "therapeutically effective amount" means thetotal amount of each active component of the pharmaceutical compositionor method that is sufficient to show a meaningful patient benefit, e.g.,amelioration of symptoms of, healing of, or increase in rate of healingof such conditions. When applied to an individual active ingredient,administered alone, the term refers to that ingredient alone. Whenapplied to a combination, the term refers to combined amounts of theactive ingredients that result in the therapeutic effect, whetheradministered in combination, serially or simultaneously.

In practicing the method of treatment or use of the present invention, atherapeutically effective amount of CTLA-8 protein is administered to amammal. CTLA-8 protein may be administered in accordance with the methodof the invention either alone or in combination with other therapiessuch as treatments employing cytokines, lymphokines or otherhematopoietic factors. When co-administered with one or more cytokines,lymphokines, other hematopoietic factors or vaccine components (such asantigens or other adjuvants), CTLA-8 protein may be administered eithersimultaneously with the cytokine(s), lymphokine(s), other hematopoieticfactor(s), thrombolytic or anti-thrombotic factors, or sequentially. Ifadministered sequentially, the attending physician will decide on theappropriate sequence of administering CTLA-8 protein in combination withcytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolyticor anti-thrombotic factors.

Administration of CTLA-8 protein used in the pharmaceutical compositionor to practice the method of the present invention can be carried out ina variety of conventional ways, such as oral ingestion, inhalation, orcutaneous, subcutaneous, or intravenous injection. Intravenousadministration to the patient is preferred.

When a therapeutically effective amount of CTLA-8 protein isadministered orally, CTLA-8 protein will be in the form of a tablet,capsule, powder, solution or elixir. When administered in tablet form,the pharmaceutical composition of the invention may additionally containa solid carrier such as a gelatin or an adjuvant The tablet, capsule,and powder contain from about 5 to 95% CTLA-8 protein, and preferablyfrom about 25 to 90% CTLA-8 protein. When administered in liquid form, aliquid carrier such as water, petroleum, oils of animal or plant originsuch as peanut oil, mineral oil, soybean oil, or sesame oil, orsynthetic oils may be added. The liquid form of the pharmaceuticalcomposition may further contain physiological saline solution, dextroseor other saccharide solution, or glycols such as ethylene glycol,propylene glycol or polyethylene glycol. When administered in liquidform, the pharmaceutical composition contains from about 0.5 to 90% byweight of CTLA-8 protein, and preferably from about 1 to 50% CTLA-8protein.

When a therapeutically effective amount of CTLA-8 protein isadministered by intravenous, cutaneous or subcutaneous injection, CTLA-8protein will be in the form of a pyrogen-free, parenterally acceptableaqueous solution. The preparation of such parenterally acceptableprotein solutions, having due regard to pH, isotonicity, stability, andthe like, is within the skill in the art. A preferred pharmaceuticalcomposition for intravenous, cutaneous, or subcutaneous injection shouldcontain, in addition to CTLA-8 protein an isotonic vehicle such asSodium Chloride Injection, Ringer's Injection, Dextrose Injection,Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, orother vehicle as known in the art. The pharmaceutical composition of thepresent invention may also contain stabilizers, preservatives, buffers,antioxidants, or other additive known to those of skill in the art.

The amount of CTLA-8 protein in the pharmaceutical composition of thepresent invention will depend upon the nature and severity of thecondition being treated, and on the nature of prior treatments which thepatient has undergone. Ultimately, the attending physician will decidethe amount of CTLA-8 protein with which to treat each individualpatient. Initially, the attending physician will administer low doses ofCTLA-8 protein and observe the patient's response. Larger doses ofCTLA-8 protein may be administered until the optimal therapeutic effectis obtained for the patient, and at that point the dosage is notgenerally increased further. It is contemplated that the variouspharmaceutical compositions used to practice the method of the presentinvention should contain about 0.1 μg to about 100 mg of CTLA-8 proteinper kg body weight, preferably about 0.1 pg to about 10 mg of CTLA-8protein per kg body weight, more preferably about 0.1 μg to about 100 μgof CTLA-8 protein per kg body weight, most preferably preferably about0.1 μg to about 10 μg of CTLA-8 protein per kg body weight.

The duration of intravenous therapy using the pharmaceutical compositionof the present invention will vary, depending on the severity of thedisease being treated and the condition and potential idiosyncraticresponse of each individual patient. It is contemplated that theduration of each application of the CTLA-8 protein will be in the rangeof 12 to 24 hours of continuous intravenous administration. Ultimatelythe attending physician will decide on the appropriate duration ofintravenous therapy using the pharmaceutical composition of the presentinvention.

CTLA-8 protein of the invention may also be used to immunize animals toobtain polyclonal and monoclonal antibodies which specifically reactwith the CTLA-8 protein and which may inhibit CTLA-8 binding to itsreceptor. Such antibodies are also useful for performing diagnosticsassays for CTLA-8 in accordance with known methods. Such antibodies maybe obtained using the entire CTLA-8 protein as an immunogen, or by usingfragments of human CTLA-8 protein. The peptide immunogens additionallymay contain a cysteine residue at the carboxyl terminus, and areconjugated to a hapten such as keyhole limpet hemocyanin (KLH).Additional peptide immunogens may be generated by replacing tyrosineresidues with sulfated tyrosine residues. Methods for synthesizing suchpeptides are known in the art, for example, as in R. P. Merrifield, J.Amer. Chem. Soc. 85, 2149-2154 (1963); J. L. Krstenansky, et al., FEBSLett. 211, 10 (1987).

Neutralizing or non-neutralizing antibodies (preferably monoclonalantibodies) binding to human CTLA-8 protein may also be usefulltherapeutics for certain tumors and also in the treatment of conditionsdescribed above. These neutralizing monoclonal antibodies are capable ofblocking the ligand binding to the human CTLA-8 protein or mayh promoteclearance of protein from the patient.

Because of their homology to human CTLA-8, rat CTLA-8 proteins, herpesCTLA-8 proteins and IL17 proteins (the "human CTLA-8" of Golstein etal., supra) will also possess CTLA-8 activity as described above. As aresult, rat and herpes CTLA-8 proteins and IL-17 proteins, as well asactive fragments and variants thereof, can be used in preparation ofpharmaceutical compositions and in methods of treatment as described forhuman CTLA-8. Rat and herpes CTLA-8 proteins, and active fragments andvariants thereof, can be produced as described above using thepolynucleotides (or fragments or variants thereof) described in SEQ IDNO:3 and SEQ ID NO:5, respectively. Rat and herpes CTLA-8 may also beproduced as described in Rouvier et al., J. Immunol. 1993, 150,5445-5456. CTLA-8 proteins of other species can also be used asdescribed herein. cDNAs encoding rat CTLA-8 and herpes CTLA-8 weredeposited with the American Type Culture Collection on Jul. 6, 1995 andassigned accession numbers ATCC 69867 and ATCC 69866, respectively.IL-17 proteins may also be produced as described in Golstein et al.,supra.

Because of its homology to IL-17, the human CTLA-8 (B18) proteins of thepresent invention may also share some activities with IL-17.

For the purposes of treatment or therapy, any of the proteins discussedor disclosed herein may be administered by in vivo expression of theprotein in a mammalian subject. In such instances, a polynucleotideencoding the desired protein is administered to the subject in mannerallowing expression in accordance with known methods, including withoutlimitation the adenovirus methods disclosed herein.

EXAMPLE 1 Isolation of Human CTLA-8 cDNA

A partial clone for human CTLA-8 was isolated from a cDNA library madefrom RNA isolated from stimulated human peripheral blood mononuclearcells. This partial was identified as "B 18." B 18 is sometimes usedherein to refer to the human CTLA-8 of the present invention. Homologysearches identified this partial clone as being related to the herpesand rat CTLA-8 genes. DNA sequence of this partial clone was used toisolate the full-length clone.

In order to isolate a full-length cDNA for B 18, a directional,full-length cDNA library by standard means in the COS expression vectorpMV2. The cDNA library was transformed into E. coli by electroporation.The bulk of the original transformed cDNA library was frozen in glycerolat -80° C. An aliquot was titered to measure the concentration oftransformed E. coli. The E. coli were thawed, diluted to 76,000/0.1 mlin media containing ampicillin, and 0.1 ml was distributed into thewells of a microtiter dish in an 8×8 array. The microtiter dish wasplaced at 37° C. overnight to grow the E. coli.

To prepare DNA for PCR, 20 μl aliquots of culture from each well werewithdrawn and pooled separately for each row and column of eight wells,giving 16 pools of 160 μl each. The E. coli were pelleted, resuspendedin 160 μl of standard lysis buffer consisting of 10 mM TrisHCI pH8, 1 mMEDTA, 0.01% Triton X-100, and lysed by heating to 95° C. for 10 minutes.

To identify which of the wells contained E. coli transformed with B 18,PCR was performed first on the DNA preps corresponding to the eightcolumns. The PCR consisted of two sequential reactions with nestedoligonucleotides using standard conditions. The oligonucleotides usedfor the PCR reaction were derived from the sequence of the partial B 18clone. They were:

B185: CACAGGCATACACAGGAAGATACATTCA (SEQ ID NO:7)

B183: TCTTGCTGGATGGGAACGGAATTCA (SEQ ID NO:8)

B18N: ATACATTCACAGAAGAGCTTCCTGCACA (SEQ ID NO:9)

The PCR conditions were 2.5 mM MgCl₂ and 95° C.×2 min for one cycle, 95°C.×1 min plus 68° C.×1 mnin for 30 cycles, and 68° C.×10 for one cycle.Each reaction was 20 μl. The first reaction contained oligonucleotidesB185 and B183 and 1 μl of the DNA preparations. The second reactioncontained oligonucleotides B183 and B18N and 1 μl of the first reaction.

DNA preps that potentially contained a full-length B18 cDNA clone wereidentified by agarose gel electrophoresis on an aliquot of the secondPCR reaction. A DNA band of the correct mobility was assumed to bederived from a B18 cDNA. Next the same sequence of PCR reactions and gelanalysis was done on the DNA preps corresponding to the eight rows. Theintersection of a row and a column identified well A2 as potentiallycontaining B18, narrowing it down to the 76,000 E. coli originallyseeded into that well.

To further purify the individual E. coli containing the putativefull-length B18 cDNA clone, the concentration of E. coli in well A2 wasmeasured by titering and plating dilutions of the well. Then 7600 E.coli were seeded into the wells of a second microtiter plate in an 8×8array. The E. coli were grown overnight; wells were pooled, and DNA wasprepared as described above. To identify which of these wells containedE. coli transformed with B18, sequential PCR reactions were performedessentially as described above. Agarose gel electrophoresis identifiedwell B2 as potentially containing a B18 cDNA.

The E. coli containing this cDNA was further purified by seeding wellsof a microtiter plate with 253 E. coli per well and proceeding as forthe purification of the E. coli in well A2. Well C3 was identified ascontaining a putative full-length B18 cDNA clone. The exact E. coli wasidentified by plating the contents of the well onto bacterial culturemedia and then screening the E. coli colonies following establishedprotocols. The probe for these hybridizations was a PCR fragmentgenerated by doing a PCR reaction on the B18 clone using as primers theoligonucleotides described above (SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9). When a single colony was identified, DNA was prepared andsequenced by standard methods. Comparison of this sequence to thesequence of the original partial clone confirmed identity and that theisolated cDNA was full-length.

The full-length clone was deposited with the American Type CultureCollection on Jul. 6, 1995 and assigned accession number ATCC 69868.

EXAMPLE 2 Expression of Human CTLA-8

The full-length B18 clone for human CTLA-8 was transfected into COScells which were then labelled with ³⁵ S-methionine. An aliquot ofconditioned medium from the transfected cell culture was reduced,denatured and electrophoresed on polyacrylamide gels. Autoradiographs ofthose gels are reproduced in FIG. 2. The band indicated by the arrowdemonstrates expression of human CTLA-8.

EXAMPLE 3 Inhibition of Angiogenesis by Human CTLA-8

The ability of human CTLA-8 to inhibit angiogenesis was examined in anangiostatic activity assay (endothelial cell proliferation assay). Theassay was done in a 96 well plate. Primary human umbilical cells(HUVECs) were seeded to 2×10³ cells per well in EGM medium(Clonetics)/20% FCS and incubated at 37° C. for 24 hr. The cells werethen starved in M199 medium (GIECO BRL) containing 10% charcoal treatedserum (M199-CS) for 48 hr at 37° C. Conditioned media containing B18(human CTLA-8) was obtained from transfected COS or stably expressingCHO cells and 1:10, 1:50, 1:250, and 1:1250 dilutions prepared inM199-CS medium containing 100 ng/ml FGF. The dilutions ofB18 were addedto the starved cells and incubated for 72 hr at 37° C. The cells werethen radiolabeled by [³ H]-thymidine for 6 hr. Radiolabeled cells werewashed with PBS and trypsinized for liquid scintilation counting.Results were plotted using Kaleidograph software. The results are shownin FIG. 3. In the figure, "Med" is the mock control, "B18" and "B18-1"were conditioned medium from two independent transfections of COS withDNA encoding human CTLA-8 (B18). IFNγ was used as a positive controlangiostatic (i.e., angiogenesis inhibition) activity. These datademonstrate that human CTLA-8 (B18) inhibits angiogenesis.

EXAMPLE 4 Hematopoietic Activity of Human CTLA-8

The hematopoietic activity of human CTLA-8 (B118) expressed in vivo wasexamined by construction of a recombinant adenovirus vector.

The B18 cDNA in the expression plasmid Adori 2-12 B18 was driven by thecytomegalovirus(CMV) immediate early promoter and enhancer.

The Adori 2-12 vector was created by addition of an SV40 origin andenhancer to a known adenovirus vector (Barr et al., Gene Therapy 1:51(1994); Davidson et al., Nature Genetics 3:219 (1993)). TheHindIII/BamHI fragment encoding the SV40 origin and enhancer wasisolated from the pMT2 mammalian expression vector, blunted with Klenowand cloned into the Nati site (blunted with Klenow) of the Ad5expression vector.

The vector was derived by digesting pNOT-B18 cDNA with Sal, filling inthe 5' overhang with Klenow to generate a blunt end and digesting withEcoRI to isolate the B18 cDNA. The blunted- EcoRI B18 fragment wasinserted into the restriction sites EcoRV-EcoRI of the adenovirus vectorAdori 2-12. The CMV-B18 expression cassette was located downstream ofthe SV40 origin and enhancer, and 0-1 map units of the left hand end ofthe adenovirus type 5(Ad5). The SV40 splice donor and acceptor werelocated between the CMV promoter and B18 cDNA. Following the insert wasSV40 poly A site, 9-16 map units of Ad5 and the puc 19 origin.

A recombinant adenovirus was generated by homologous recombination in293 cells. AscI linearized Adori 2-12 B18 and ClaI digested AdCMVlacZwere introduced into the 293 cells using lipofectamine. Recombinantadenovirus virus was isolated and amplified on 293 cells. The virus wasreleased from infected 293 cells by three cycles of freeze-thawing. Thevirus was further purified by two cesium chloride centrifugationgradients and dialyzed against PBS 4° C. Following dialysis of the virusglycerol was added to a concentration of 10% and the virus was stored at-70° C. until use. The virus was characterized by expression of thetransgene, plaque forming units on 293 cells, particles/ml and Southernanalysis of the virus.

A single dose of 5×10¹⁰ particles of recombinant adenovirus encoding B18was injected into the tail vein of male C57/b16 mice, age 7-8 weeks.Control mice received an adenovirus encoding B-galactosidase. Four micefrom each experimental group were killed on day 7 and 14. Blood wascollected and automated hematologic analysis was performed using a Baker9000. Differential counts were performed on blood smears. Tissue washarvested, fixed in formalin, and stained with hematoxylin and eosin forhistopathology. In the first set of experiments, serum and tissues wereanalyzed 7 and 14 days post injection. A slight increase in peripheralplatelet counts were observed. The animals that received B18 exhibited aslight increase in spleen size. Macroscopic analysis of the spleenshowed an increase in splenic extramedullary hematopoiesis on day 7compared to the control. These results showed a hematopoietic growthactivity associated with B18.

In a second set of experiments 5×10¹⁰ particles of recombinantadenovirus encoding B18 were injected into the tail vein of male C57/b16mice, age 17-18 weeks. Control mice received an adenovirus encodingB-galactosidase. Blood samples were collected via retro-orbital sinus ondays 2, 5, 7, 10, 14, and 21. The hematologic analyses were performed onthe Baker 9000 automated cell counter with murine-specific settings.Analyses included WBC, RBC, HCT and PLT counts. Blood smears wereprepared and stained with Wright-Geimsa for WBC differentials based on a100 cellcount. Reticulocytes and reticulated platelets were quantitatedusing flow cytometry. Four mice from each group were killed on days 7,14, and 21. In addition to peripheral blood analysis, serum wascollected via cardiac puncture for quantitation of systemic 1-6 using acommercial kit (Endogen). Spleen and liver were collected forhistopathology, spleen and bone marrow hematopoietic progenitors werequantitated, and bone marrow smears were prepared and stained withWright-Geimsa for cell counts.

Administration of adenovirus encoding B18 resulted in a marked increasein peripheral blood neutrophils and WBC (FIG. 4). Maximum increases inneutrophils were observed at day 5 and day 7. The control mice showedlittle difference at day 5 and day 7. Peripheral blood neutrophils weresimilar in the control mice and mice that received B18 at day 21. Inboth the B18 and control groups an increase in white blood cells wasalso observed. The mice that received B18 had a greater increase in WBCbetween day 2 and day 7. By Day 21 a more pronounced increase wasobserved in the B-gal group. No other changes in cellular chemistrieswere observed (Table I).

Bone marrow cellularity was calculated from pooled femurs in each group(Table III). No significant differences were observed in either group.No significant changes were observed in bone marrow hematopoieticprogenitors from day 7, 14, and 21. The CFU-GM, BFU-E and CFU-MEG in theB18 mice were similar to the B-gal control (Table II).

Administration of the adenovirus encoding B18 resulted in an increase inCFU-GM (myeloid) and BFU-E (erythroid) progenitors in the spleencompared to animals that received the B-gal virus on day 7. The increasein progenitors in the B18 mice was 11-fold in CFU-GM and a 52-fold inBFU-E (Table II). There was a 2-fold increase in CFU-MEG at day 7 forthe B18 mice. By day 21 no significant differences were observed insplenic CFU-MEG or BFU-E between the groups (Table II). A 3-folddecrease in CFU-GM was observed in mice that received adenovirusencoding B18. A slight increase in spleen size at day 7 was observed inthe B18 group. This is consistent with an increase in spleniccellularity. By day 14 and day 21 spleen weights were similar to thecontrol group (Table III). Macroscopic analysis of the spleen showed anincrease in splenic extramedullary hematopoiesis of the B18 mice on day7 compared to the control.

The bone marrow myeloid: erythroid ratios (Table IV) suggest agranulocytic hyperplasia with a possible erythroid hypoplasia in micethat received adenovirus B18 on day 7. By day 21 the ratio in the B-galgroup was higher. No changes were observed in the IL6 serum levels.

These results show a hematopoietic activity associated with theadministration of adenovirus encoding B18 (human CTLA-8). Increases inneutrophils and white blood cells were observed at day 7 in animals thatreceived B18 adenovirus. The data showed that B18 resulted in increasein splenic CFU-GM and BFU-E 7 days post administration compared to thecontrol animals. Splenic extramedullary hematopoiesis on day 7 supportthat B18 exhibits a hematopoietic growth activity. These data suggestthat B18 may moblize early hematopoietic precursors.

                                      TABLE I                                     __________________________________________________________________________    Peripheral hematology for day 2, 5, 7, 10, 14, and 21.                        __________________________________________________________________________    Study A54-4B . . . B18 (Platelets) Day 2 . . . 4-25-96.                            WBC                                                                                                                                  ×10                                                                   Neuts ANC                                                                     Lymphs ALC Eos                                                                Monos RBC                                                                     Retics Abs                                                                    Relics HCT PLT                                                                RPlt Abs RPlt                                                                  Group A 3/uL %                                                               ×10 3/uL                                                                % ×10                                                                   3/uL % %                                                                      ×10 6/uL                                                                % ×10                                                                   6/uL % ×10                                                               3/uL %                                                                       ×10           __________________________________________________________________________                                                              3/uL                B-Gal #1                                                                           5.4 46 2.16 54  2.62 0  6   10.66                                                                              3.65                                                                              0.46 48.0                                                                             836  11.94                                                                            99.82                 B-Gal #2 7.4 25 1.85 65 4.81 3 7 12.34 2.04 0.25 56.6 900 10.10 90.90                                                                  B-Gal #3 6.6                                                                 46 2.72 52 3.54                                                               2 6 11.26 3.26                                                                0.37 51.6 894                                                                 9.77 87.34                                                                     B-Gal #4 6.6                                                                 23 2.02 64 5.63                                                               1 12 12.00 2.55                                                               0.31 54.8 840                                                                 10.63 89.29                                                                    AVG 7.1 32.0                                                                 2.19 58.8 4.22                                                                1.5 7.8 11.62                                                                 2.88 0.33 52.8                                                                868 10.61 91.84       SEM 0.7 4.6 0.19 3.4 0.61 6.6 1.4 0.33 0.36 0.03 1.9  17 0.48 2.76                                                                     B18 #1 11.4 59                                                               6.73 31 3.53 1                                                                9 11.16 4.88                                                                  0.54 52.4 1242                                                                14.92 185.31                                                                   B18 #2 9.2 30                                                                2.76 62 5.70 3                                                                5 10.14 3.97                                                                  0.40 48.0 632                                                                 10.90 68.89                                                                    B18 #3 5.0 51                                                                2.55 40 2.00 0                                                                9 11.16 3.23                                                                  0.36 51.2 832                                                                 11.18 93.02                                                                    B18 #4 6.4 41                                                                2.62 55 3.52 0                                                                4 16.66 3.09                                                                  0.33 49.2 904                                                                 17.31 156.48                                                                   AVG 6.0 45.3                                                                 3.67 47.0 3.69                                                                1.0 6.8 10.62                                                                 3.79 0.41 50.2                                                                903 13.58                                                                     125.92                SEM 1.4 6.3 1.02 7.0 0.76 0.7 1.3 0.24 0.41 0.05 1.0 127 1.55 27.07         __________________________________________________________________________    Study A54-4B . . . B18 (Platelets) Day 5 . . . 4-28-96.                            WBC                                                                         ×10   Neuts ANC Lymphs ALC Eos Monos RBC Retics Abs Relics HCT                                                                 PLT RPlt Abs                                                                  RPlt                  Group B 3/uL % ×10 3/uL % ×10 3/uL % % ×10 6/uL %                                                                   ×10 6/uL                                                                % ×10                                                                   3/uL % ×10                                                               3/uL               __________________________________________________________________________    B-Gal #1                                                                           7.6 14 1.06 78  5.93 3  5   11.26                                                                              5.25                                                                              0.59 52.4                                                                             1082 15.51                                                                            167.82                B-Gal #2 10.6 20 2.12 78 8.27 1 1 10.72 4.71 0.50 49.4  994 17.37                                                                     172.66                B-Gal #3 8.8 18 1.51 69 6.07 2 11 11.12 3.40 0.38 51.2  916 9.55 87.48                                                                 B-Gal #4 10.8                                                                38 4.10 58 6.26                                                               0 4 10.22 6.21                                                                0.63 47.0 1092                                                                13.93 152.12                                                                   AVG 9.5 22.5                                                                 2.20 70.8 6.63                                                                1.5 5.3 10.83                                                                 4.89 0.53 50.0                                                                1021 14.09                                                                    145.02                SEM 0.8 5.3 0.67 4.8 0.55 0.6 2.1 0.23 0.59 0.06 1.2  41 1.67 19.67                                                                    B18 #1 14.8 18                                                               2.66 71 10.51 1                                                               10 12.66 2.31                                                                 0.29 57.0 1204                                                                7.57 91.14                                                                     B18 #2 14.2 37                                                               5.25 53 7.53 2                                                                8 9.80 3.32                                                                   0.33 44.6  888                                                                14.33 127.25                                                                   B18 #3 12.8 30                                                               3.84 59 7.55 1                                                                10 12.12 4.12                                                                 0.50 55.6 1134                                                                10.15 115.10                                                                   B18 #4 16.0 58                                                               9.28 37 5.92 0                                                                5 11.04 3.93                                                                  0.43 50.8 1166                                                                15.75 183.65                                                                   AVG 14.5 35.8                                                                5.26 550 7.88                                                                 1.0 8.3 11.41                                                                 3.42 0.39 52.0                                                                1098 11.95                                                                    129.28                SEM 0.7 8.4 1.44 7.1 0.96 0.4 1.2 0.63 0.41 0.05 2.8  71 1.88 19.61         __________________________________________________________________________    Study A54-4B . . . B18 (Platelets) Day 7 . . . 4-40-96.                            WBC                                                                         ×10   Neuts ANC Lymphs ALC Eos Monos RBC Retics Abs Relics HCT                                                                 PLT RPlt Abs                                                                  RPlt                  Group C 3/uL % ×10 3/uL % ×10 3/uL % % ×10 6/uL %                                                                   ×10 6/uL                                                                % ×10                                                                   3/uL % ×10                                                               3/uL               __________________________________________________________________________    B-Gal #1                                                                           15.2                                                                              14 2.13 69  10.49                                                                              1  16  11.04                                                                              3.54                                                                              0.39 50.8                                                                              862 12.46                                                                            107.41                B-Gal #2 14.0 12 16.8 81 11.34 0 7 11.38 5.05 0.57 52.6 1104 14.91                                                                    164.81                B-Gal #3 14.8 14 2.07 73 10.80 1 12 10.92 5.42 0.59 49.6  952 11.49                                                                   109.38                AVG 14.7 13.3 1.96 74.3 10.88 0.7 11.7 11.11 4.67 0.52 51.0  973 12.95                                                                127.13                SEM 0.4 0.7 0.14 3.5 0.25 0.3 2.6 0.14 0.58 0.06 0.9  71 1.02 18.75                                                                    B18 #1 19.4 33                                                               6.40 62 12.03 0                                                               5 10.14 2.93                                                                  0.30 45.2  864                                                                12.80 110.59                                                                   B18 #2 25.4 39                                                               9.91 53 13.46 0                                                               8 9.48 6.05                                                                   0.57 43.6 1288                                                                12.49 160.87                                                                   B18 #3 23.6 44                                                               10.38 50 11.80                                                                0 6 9.74 5.17                                                                 0.50 44.4 1076                                                                15.41 165.81                                                                   B18 #4 12.8 15                                                               1.92 75 9.60 0                                                                10 9.54 6.26                                                                  0.60 43.4 1136                                                                15.88 180.40                                                                   AVG 20.3 32.8                                                                7.15 60.0 11.72                                                               0.0 7.3 9.72                                                                  5.10 0.49 44.2                                                                1091 14.15                                                                    154.42                SEM 2.8 6.3 1.96 5.6 0.80 0.0 1.1 0.15 0.76 0.07 0.4  88 0.87 15.19         __________________________________________________________________________    Study A54-4B . . . B18 (Platelets) Day 10 . . . 5-3-96.                            WBC                                                                         ×10   Neuts ANC Lymphs ALC Eos Monos RBC Retics Abs Relics HCT                                                                 PLT RPlt Abs                                                                  RPlt                  Group A 3/uL % ×10 3/uL % ×10 3/uL % % ×10 6/uL %                                                                   ×10 6/uL                                                                % ×10                                                                   3/uL % ×10                                                               3/uL               __________________________________________________________________________    B-Gal #1                                                                           18.8                                                                              17 3.16 69  12.83                                                                              3  11  10.22                                                                              12.41                                                                             1.27 46.8                                                                             1460 16.20                                                                            236.52                B-Gal #2 13.2 16 2.11 79 10.43 1 4 10.48 6.0 0.63 46.6 1128 14.48                                                                     163.33                B-Gal #3 19.6 16 3.14 74 14.50 0 10 10.72 6.24 0.67 49.4 1338 16.58                                                                   221.84                B-Gal #4 18.6 21 3.91 72 13.39 3 4 10.44 7.59 0.79 48.4 1068 14.35                                                                    153.26                AVG 17.5 17.5 3.08 73.5 12.79 1.8 7.3 10.47 8.06 0.84 46.4 1249 15.40                                                                 193.74                SEM 1.5 1.2 0.37 2.1 0.86 0.8 1.9 0.18 1.49 0.15 0.6  91 0.58 20.76                                                                    B18 #1 14.2 33                                                               4.69 56 7.95 5                                                                6 8.70 11.97                                                                  1.04 39.2 1760                                                                14.49 255.02                                                                   B18 #2 17.6 35                                                               6.16 57 10.03 1                                                               7 9.04 9.48                                                                   0.86 42.0 1104                                                                16.88 208.44                                                                   B18 #3 16.2 39                                                               6.32 57 9.23 1                                                                3 4.74 16.77                                                                  0.79 22.4  894                                                                29.19 260.96                                                                   B18 #4 14.2 25                                                               3.55 66 9.37 1                                                                8 9.30 9.93                                                                   0.92 42.0 1416                                                                16.81 238.03                                                                   AVG 15.6 33.0                                                                5.18 59.0 9.15                                                                2.0 6.0 7.95                                                                  12.04 0.90 36.4                                                               1294 19.84                                                                    240.61                SEM 0.8 2.9 0.66 2.3 0.43 1.0 1.1 1.08 1.67 0.05 4.7  189 3.24 11.77        __________________________________________________________________________    Study A54-4B . . . B18 (Platelets) Day 14 . . . 5-7-96.                            WBC                                                                         ×10   Neuts ANC Lymphs ALC Eos Monos RBC Retics Abs Relics HCT                                                                 PLT RPlt Abs                                                                  RPlt                  Group B 3/uL % ×10 3/uL % ×10 3/uL % % ×10 6/uL %                                                                   ×10 6/uL                                                                % ×10                                                                   3/uL % ×10                                                               3/uL               __________________________________________________________________________    B-Gal #1                                                                           17.8                                                                              18 3.20 74  13.17                                                                              0  8   10.86                                                                              5.97                                                                              0.65 50.8                                                                             1360 11.03                                                                            150.01                B-Gal #2 20.4 26 5.30 66 13.46 1 7 10.92 7.07 0.77 50.8 1616 8.18                                                                     132.19                B-Gal #3 16.0 7 1.12 90 14.40 1 3 11.36 6.41 0.73 52.8 1298 7.36 95.53                                                                 B-Gal #4 18.0                                                                36 6.48 57                                                                    10.26 1 6 9.30                                                                7.62 0.71 43.0                                                                1672 10.05                                                                    168.04                AVG 18.1 21.8 4.03 71.8 12.82 0.8 6.0 10.61 6.77 0.71 49.4 1487 9.16                                                                  136.44                SEM 0.9 6.1 1.18 7.0 0.89 0.3 1.1 0.45 0.36 0.03 2.2  93 0.84 15.48                                                                    B18 #1 15.4 9                                                                1.39 81 12.47 1                                                               9 10.62 5.74                                                                  0.61 48.2 1262                                                                9.51 120.02                                                                    B18 #2 15.4 31                                                               4.77 58 8.93 2                                                                9 9.76 10.33                                                                  1.01 44.6 1093                                                                14.29 156.05                                                                   B18 #3 13.4 42                                                               5.63 39 5.23 0                                                                19 10.34 4.99                                                                 0.52 46.6 1376                                                                15.79 217.27                                                                   B18 #4 11.6 57                                                               6.61 34 3.94 2                                                                7 9.38 5.57                                                                   0.52 43.0 1092                                                                16.66 181.93                                                                   AVG 14.0 34.8                                                                4.60 53.0 7.64                                                                1.3 11.0 10.03                                                                6.66 0.66 45.6                                                                1206 14.06                                                                    168.82                SEM 0.9 10.1 1.14 10.7 1.93 0.5 2.7 0.28 1.23 0.12 1.1  70 1.59             __________________________________________________________________________                                                              20.54               Study A54-4B . . . B18 (Platelets) Day 21 . . . 5-14-96.                           WBC                                                                         ×10   Neuts ANC Lymphs ALC Eos Monos RBC Retics Abs Relics HCT                                                                 PLT RPlt Abs                                                                  RPlt                  Group B 3/uL % ×10 3/uL % ×10 3/uL % % ×10 6/uL %                                                                   ×10 6/uL                                                                % ×10                                                                   3/uL % ×10                                                               3/uL               __________________________________________________________________________    B-Gal #1                                                                           25.4                                                                              23 5.84 67  17.02                                                                              0  10  9.22 8.15                                                                              0.75 42.8                                                                             1776 9.61                                                                             170.67                B-Gal #2 19.6 19 3.72 69 13.52 0 12 9.50 9.95 0.95 44.4 1652 9.44                                                                     156.89                B-Gal #3 27.6 11 3.04 82 22.63 3 4 9.74 8.84 0.66 45.8 1684 11.45                                                                     192.82                B-Gal #4 28.0 13 3.64 83 23.24 0 4 9.04 7.54 0.68 41.6 1346 10.48                                                                     141.06                AVG 25.2 16.4 4.06 75.3 19.10 0.8 7.5 9.36 8.62 0.81 43.7 1617 10.25                                                                  165.36                SEM 1.9 2.8 0.61 4.2 2.33 0.6 2.1 0.15 0.52 0.06 0.9  94 0.46 10.97                                                                    B18 #1 18.6 9                                                                1.67 83 15.44 1                                                               7 9.84 7.40                                                                   0.73 43.8 1642                                                                8.37 137.44                                                                    B18 #2 16.2 48                                                               7.78 45 7.29 1                                                                6 11.10 7.97                                                                  0.88 51.4 1970                                                                10.79 212.37                                                                   B18 #3 14.8 53                                                               7.84 42 6.22 0                                                                5 7.52 20.26                                                                  1.52 38.0 1568                                                                10.50 164.64                                                                   B18 #4 15.2 18                                                               2.74 74 11.25 2                                                               6 9.64 7.02                                                                   0.68 43.0 1422                                                                7.60 108.07                                                                    AVG 16.2 32.0                                                                5.01 61.0 10.05                                                               1.0 6.0 9.53                                                                  10.66 0.95 44.1                                                               1651 9.31                                                                     155.63                SEM 0.9 10.9 1.63 10.3 2.10 0.4 0.4 0.74 3.21 0.20 2.6  116 0.78            __________________________________________________________________________                                                              22.16           

                  TABLE II                                                        ______________________________________                                        Bone marrow and Splenic Hematopoietic Progenitors                               Bone     CFU-MEG      CFU-GM     BFU-E                                      Marrow*                                                                              B-Gal   B18      B-Gal B18    B-Gal B18                                ______________________________________                                        Day 7  16.0 ±                                                                             15.7 ±                                                                              307   241 ± 78                                                                          51 ± 19                                                                          25 ± 11                            3.5 3.1 ± 117                                                             Day 14 10.7 ± 15.3 ± 233 ± 373 ± 35 30 ± 10 60 ± 30                                                    2.3 1.2 15                         Day 21 5.7 ± 6.7 ± 3.1 170 ± 160 ± 27 40 ± 10 27 ± 6                                                   0.6  17                            Spleen**                                                                      Day 7 9.3 ± 19.5 ± 27 ± 3 298 ± 6 1.3 ± 68 ± 10                                                        1.6 1.5   1.2                      Day 14 9.7 ± 12.7 ± 267 ± 197 ± 21 33 ± 6 10 ± 10                                                      0.6 0.6 32                         Day 21 17.0 ± 19.3 ± 187 ± 6 73 ± 15 23 ± 6 23 ± 6                                                     1.0 2.5                          ______________________________________                                    

Hematopoietic precursors were determined form pooled spleen and bonemarrow samples from four animals in each group. For quantitation ofCFU-GM and BFU-E, either 1×10⁴ bone marrow cells or 1×10⁵ spleen cellswere added to complete alpha methylcelluose medium (0.9% methylcellulosein alpha medium, 30% fetal bovine serum, 1% bovine serum albumin, 10-4M2-mercaptoethanol, 2 mM L-glutamine, 2% murine spleen cell conditionedmedium, and 3 U/mL erythropoietin) and aliquoted into 35 mm tissueculture dishes in a final volume of 1.0 mL. Cultures were incubated for7 days at 37° C., and 5% CO₂. Microscopic colonies were defined asclusters of 50 or more cells. For quantitaion of CFU-MEG, either 1×10⁵bone marrow cells or 1×10⁶ spleen cells were added to complete alphamethylcellulose medium and incubated as described above. Megakaryocytecolonies were defined as a group of 3 or more cells.

*Bone marrow progenitors are represented as mean±sd number of coloniesper 10⁵ cells.

**Spleen progenitors are represented as mean±sd number of colonies per10⁶ cells.

                  TABLE III                                                       ______________________________________                                        Spleen Weights and Femur Cellularity                                                                       Femur                                              Spleen Wt.   Cellularity                                                      (Mg) B-Gal B18 (× 10.sup.6) B-Gal B18                                 ______________________________________                                        Day 7    187 ± 19                                                                            224 ± 29                                                                            Day 7  28     23                                     Day 14 175 ± 13 170 ± 10 Day 14 28 27                                   Day 21 174 ± 21 151 ± 27 Day 21 28 26                                 ______________________________________                                    

Spleen weights were determined at time of sacrifice are represented asmeans±sd from four animals.

                  TABLE IV                                                        ______________________________________                                        Bone Marrow Myeloid:Erythoid Ratios                                               Group    Mouse #  Day 7     Day 14                                                                              Day 21                                  ______________________________________                                        B-gal    1        1.43        2.12  5.78                                         2 0.91 2.46 5.83                                                              3 1.62 1.03 3.66                                                              4  5.44 4.82                                                                 AVG  1.32 2.76 5.02                                                           SD  0.37 0.37 1.89                                                            B-gal 1 5.59 2.01 2.02                                                         2 6.51 1.25 2.13                                                              3 5.49 1.58 1.81                                                              4 0.50 2.51 2.92                                                             AVG  4.52 1.86 2.22                                                           SD  1.29 2.72 0.56                                                          ______________________________________                                    

All entries represent the number of myeloid cells per 1 erythroid cell.Normal mouse ratios are approximately 1:1 to 2:1.

EXAMPLE 5 Additional Experiments Relating to Hematopoietic Activity ofHuman CTLA-8

B18 (human CTLA-8) was tested for the ability to induce production offactors having hematopoietic activity in a factor-dependent cellproliferation assay using the human erythroleukemic cell line, TF-1(Kitamura et al., J. Cell Physiol. 140:323 (1989)). The cells wereinitially grown in the presence of rhGMCSF (100 U/ml). The cells werefed three days prior to setting up the assay. The assay conditions wereas follows:

    ______________________________________                                        cells/well             5000/200 μl                                           incubation time 3 days                                                        pulse time 4 hours                                                            amount of tritiated thymidine 0.5 μCi/well                                 counting time 1 minute                                                        replicates 2                                                                ______________________________________                                    

B18 alone, conditioned medium (CM) from B18 induced HS-5 cells wereassayed. Buffer alone, CM from HS-5 cells induced with buffer and CMfrom uninduced HS-5 cells were assayed as controls. Results are shown inFIG. 5. B18 (human CTLA-8) demonstrated an abilit to induce productionof factors which induced TF-1 proliferation. This activity wassubstantially eliminated by the addition of anti-GMCSF antibodies. Thesedata demonstrate that human CTLA-8 (B18) is able to inducehematopoiesis. Particularly, without being bound by any theory, itappears that human CTLA-8 (B18) induces production of GM-CSF and/orIL-3.

EXAMPLE 6 Ability of Human CTLA-8 to Induce Production of IL-6 and IL-8

MRC5 cells were incubated in the presence of human CTLA-8 (B18) andproduction of IL-6 and IL-8 were measured. Herpes CTLA-8 (IL-17) wasused as a positive control. Applicants' human CTLA-8 (B18) demonstratedtitratable production of both IL-6 and IL-8 (see FIGS. 6 and 7).

All patent and literature references cited herein are incorporated byreference as if fully set forth.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 9                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 813 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 56..544                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - -   GGGAAGATAC ATTCACAGAA AGAGCTTCCT GCACAAAGTA AGCCACCA - #GC GCAAC     ATG    58                                                                                        - #                  - #                  - #              Met                                                                                               - #                  - #                  - #               1                                                                             - -   ACA GTG AAG ACC CTG CAT GGC CCA GCC A - #TG GTC AAG TAC TTG CTG       CTG    106                                                                        Thr Val Lys Thr Leu His Gly Pro Ala M - #et Val Lys Tyr Leu Leu Leu                        5  - #                10  - #                15                - -   TCG ATA TTG GGG CTT GCC TTT CTG AGT G - #AG GCG GCA GCT CGG AAA       ATC    154                                                                        Ser Ile Leu Gly Leu Ala Phe Leu Ser G - #lu Ala Ala Ala Arg Lys Ile                   20       - #           25       - #           30                    - -   CCC AAA GTA GGA CAT ACT TTT TTC CAA A - #AG CCT GAG AGT TGC CCG       CCT    202                                                                        Pro Lys Val Gly His Thr Phe Phe Gln L - #ys Pro Glu Ser Cys Pro Pro               35           - #       40           - #       45                        - -   GTG CCA GGA GGT AGT ATG AAG CTT GAC A - #TT GGC ATC ATC AAT GAA       AAC    250                                                                        Val Pro Gly Gly Ser Met Lys Leu Asp I - #le Gly Ile Ile Asn Glu Asn           50               - #   55               - #   60               - #        65                                                                               - -   CAG CGC GTT TCC ATG TCA CGT AAC ATC G - #AG AGC CGC TCC ACC TCC      CCC    298                                                                        Gln Arg Val Ser Met Ser Arg Asn Ile G - #lu Ser Arg Ser Thr Ser Pro                          - # 70                 - # 75                 - # 80         - -   TGG AAT TAC ACT GTC ACT TGG GAC CCC A - #AC CGG TAC CCC TCG GAA       GTT    346                                                                        Trp Asn Tyr Thr Val Thr Trp Asp Pro A - #sn Arg Tyr Pro Ser Glu Val                       85   - #               90   - #               95                - -   GTA CAG GCC CAG TGT AGG AAC TTG GGC T - #GC ATC AAT GCT CAA GGA       AAG    394                                                                        Val Gln Ala Gln Cys Arg Asn Leu Gly C - #ys Ile Asn Ala Gln Gly Lys                  100        - #         105        - #         110                    - -   GAA GAC ATC TCC ATG AAT TCC GTT CCC A - #TC CAG CAA GAG ACC CTG       GTC    442                                                                        Glu Asp Ile Ser Met Asn Ser Val Pro I - #le Gln Gln Glu Thr Leu Val              115            - #     120            - #     125                        - -   GTC CGG AGG AAG CAC CAA GGC TGC TCT G - #TT TCT TTC CAG TTG GAG       AAG    490                                                                        Val Arg Arg Lys His Gln Gly Cys Ser V - #al Ser Phe Gln Leu Glu Lys          130                - # 135                - # 140                - #       145                                                                              - -   GTG CTG GTG ACT GTT GGC TGC ACC TGC G - #TC ACC CCT GTC ATC CAC      CAT    538                                                                        Val Leu Val Thr Val Gly Cys Thr Cys V - #al Thr Pro Val Ile His His                          - #150                 - #155                 - #160         - -   GTG CAG TAAGAGGTGC ATATCCACTC AGCTGAAGAA GCTGTAGA - #AA TGCCACTCCT         594                                                                         Val Gln                                                                      - -   TACCCAGTGC TCTGCAACAA GTCCTGTCTG ACCCCCAATT CCCTCCAC - #TT            CACAGGACTC  654                                                                  - -   TTAATAAGAC CTGCACGGAT GGAAACAGAA AATATTCACA ATGTATGT - #GT           GTATGTACTA  714                                                                  - -   CACTTTATAT TTGATATCTA AAATGTTAGG AGAAAAATTA ATATATTC - #AG           TGCTAATATA  774                                                                  - -   ATAAAGTATT AATAATTTAA AAATAAAAAA AAAAAAAAA    - #                      - #   813                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 163 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - -   Met Thr Val Lys Thr Leu His Gly Pro A - #la Met Val Lys Tyr Leu       Leu                                                                                 1              - # 5                 - # 10                 - # 15        - -   Leu Ser Ile Leu Gly Leu Ala Phe Leu S - #er Glu Ala Ala Ala Arg       Lys                                                                                            20   - #               25   - #               30               - -   Ile Pro Lys Val Gly His Thr Phe Phe G - #ln Lys Pro Glu Ser Cys       Pro                                                                                        35       - #           40       - #           45                    - -   Pro Val Pro Gly Gly Ser Met Lys Leu A - #sp Ile Gly Ile Ile Asn      Glu                                                                                    50           - #       55           - #       60                        - -   Asn Gln Arg Val Ser Met Ser Arg Asn I - #le Glu Ser Arg Ser Thr      Ser                                                                                65               - #   70               - #   75               - #       80                                                                              - -   Pro Trp Asn Tyr Thr Val Thr Trp Asp P - #ro Asn Arg Tyr Pro Ser       Glu                                                                                               - # 85                 - # 90                 - # 95        - -   Val Val Gln Ala Gln Cys Arg Asn Leu G - #ly Cys Ile Asn Ala Gln       Gly                                                                                           100    - #             105    - #             110               - -   Lys Glu Asp Ile Ser Met Asn Ser Val P - #ro Ile Gln Gln Glu Thr       Leu                                                                                       115        - #         120        - #         125                    - -   Val Val Arg Arg Lys His Gln Gly Cys S - #er Val Ser Phe Gln Leu      Glu                                                                                   130            - #     135            - #     140                        - -   Lys Val Leu Val Thr Val Gly Cys Thr C - #ys Val Thr Pro Val Ile      His                                                                               145                - # 150                - # 155                - #      160                                                                              - -   His Val Gln                                                             - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 461 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 6..455                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - -   CCACC ATG TGC CTG ATG CTG TTG CTG CTA - #CTG AAC CTG GAG GCT ACA           47                                                                               Met Cys Leu Met Leu Leu - #Leu Leu Leu Asn Leu Glu Ala Thr                      1        - #       5           - #       10                          - -   GTG AAG GCA GCG GTA CTC ATC CCT CAA A - #GT TCA GTG TGT CCA AAC       GCC     95                                                                        Val Lys Ala Ala Val Leu Ile Pro Gln S - #er Ser Val Cys Pro Asn Ala           15               - #   20               - #   25               - #        30                                                                               - -   GAG GCC AAT AAC TTT CTC CAG AAC GTG A - #AG GTC AAC CTG AAA GTC      ATC    143                                                                        Glu Ala Asn Asn Phe Leu Gln Asn Val L - #ys Val Asn Leu Lys Val Ile                          - # 35                 - # 40                 - # 45         - -   AAC TCC CTT AGC TCA AAA GCG AGC TCG A - #GA AGG CCC TCA GAT TAC       CTC    191                                                                        Asn Ser Leu Ser Ser Lys Ala Ser Ser A - #rg Arg Pro Ser Asp Tyr Leu                       50   - #               55   - #               60                - -   AAC CGT TCC ACT TCA CCC TGG ACT CTG A - #GC CGC AAT GAG GAC CCT       GAT    239                                                                        Asn Arg Ser Thr Ser Pro Trp Thr Leu S - #er Arg Asn Glu Asp Pro Asp                   65       - #           70       - #           75                    - -   AGA TAT CCT TCT GTG ATC TGG GAG GCA C - #AG TGC CGC CAC CAG CGC       TGT    287                                                                        Arg Tyr Pro Ser Val Ile Trp Glu Ala G - #ln Cys Arg His Gln Arg Cys               80           - #       85           - #       90                        - -   GTC AAC GCT GAG GGG AAG TTG GAC CAC C - #AC ATG AAT TCT GTT CTC       ATC    335                                                                        Val Asn Ala Glu Gly Lys Leu Asp His H - #is Met Asn Ser Val Leu Ile           95               - #  100               - #  105               - #        110                                                                              - -   CAG CAA GAG ATA CTA GTC CTG AAG AGG G - #AG CCT GAG AAG TGC CCC      TTC    383                                                                        Gln Gln Glu Ile Leu Val Leu Lys Arg G - #lu Pro Glu Lys Cys Pro Phe                          - #115                 - #120                 - #125         - -   ACT TTC CGG GTG GAG AAG ATG CTG GTG G - #GC GTG GGC TGC ACC TGC       GTT    431                                                                        Thr Phe Arg Val Glu Lys Met Leu Val G - #ly Val Gly Cys Thr Cys Val                      130    - #             135    - #             140                - -   TCC TCT ATT GTC CGC CAT GCG TCC TAATAA - #                  - #              461                                                                       Ser Ser Ile Val Arg His Ala Ser                                                       145        - #         150                                           - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 150 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - -   Met Cys Leu Met Leu Leu Leu Leu Leu A - #sn Leu Glu Ala Thr Val       Lys                                                                                 1              - # 5                 - # 10                 - # 15        - -   Ala Ala Val Leu Ile Pro Gln Ser Ser V - #al Cys Pro Asn Ala Glu       Ala                                                                                            20   - #               25   - #               30               - -   Asn Asn Phe Leu Gln Asn Val Lys Val A - #sn Leu Lys Val Ile Asn       Ser                                                                                        35       - #           40       - #           45                    - -   Leu Ser Ser Lys Ala Ser Ser Arg Arg P - #ro Ser Asp Tyr Leu Asn      Arg                                                                                    50           - #       55           - #       60                        - -   Ser Thr Ser Pro Trp Thr Leu Ser Arg A - #sn Glu Asp Pro Asp Arg      Tyr                                                                                65               - #   70               - #   75               - #       80                                                                              - -   Pro Ser Val Ile Trp Glu Ala Gln Cys A - #rg His Gln Arg Cys Val       Asn                                                                                               - # 85                 - # 90                 - # 95        - -   Ala Glu Gly Lys Leu Asp His His Met A - #sn Ser Val Leu Ile Gln       Gln                                                                                           100    - #             105    - #             110               - -   Glu Ile Leu Val Leu Lys Arg Glu Pro G - #lu Lys Cys Pro Phe Thr       Phe                                                                                       115        - #         120        - #         125                    - -   Arg Val Glu Lys Met Leu Val Gly Val G - #ly Cys Thr Cys Val Ser      Ser                                                                                   130            - #     135            - #     140                        - -   Ile Val Arg His Ala Ser                                                  145                - # 150                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 459 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..453                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - -   ATG ACA TTT AGA ATG ACT TCA CTT GTG T - #TA CTT CTG CTG CTG AGC      ATA     48                                                                        Met Thr Phe Arg Met Thr Ser Leu Val L - #eu Leu Leu Leu Leu Ser Ile            1              - # 5                 - # 10                 - # 15         - -   GAT TGT ATA GTA AAG TCA GAA ATA ACT A - #GT GCA CAA ACC CCA AGA       TGC     96                                                                        Asp Cys Ile Val Lys Ser Glu Ile Thr S - #er Ala Gln Thr Pro Arg Cys                       20   - #               25   - #               30                - -   TTA GCT GCT AAC AAT AGC TTT CCA CGG T - #CT GTG ATG GTT ACT TTG       AGC    144                                                                        Leu Ala Ala Asn Asn Ser Phe Pro Arg S - #er Val Met Val Thr Leu Ser                   35       - #           40       - #           45                    - -   ATC CGT AAC TGG AAT ACC AGT TCT AAA A - #GG GCT TCA GAC TAC TAC       AAT    192                                                                        Ile Arg Asn Trp Asn Thr Ser Ser Lys A - #rg Ala Ser Asp Tyr Tyr Asn               50           - #       55           - #       60                        - -   AGA TCT ACG TCT CCT TGG ACT CTC CAT C - #GC AAT GAA GAT CAA GAT       AGA    240                                                                        Arg Ser Thr Ser Pro Trp Thr Leu His A - #rg Asn Glu Asp Gln Asp Arg           65               - #   70               - #   75               - #        80                                                                               - -   TAT CCC TCT GTG ATT TGG GAA GCA AAG T - #GT CGC TAC TTA GGA TGT      GTT    288                                                                        Tyr Pro Ser Val Ile Trp Glu Ala Lys C - #ys Arg Tyr Leu Gly Cys Val                          - # 85                 - # 90                 - # 95         - -   AAT GCT GAT GGG AAT GTA GAC TAC CAC A - #TG AAC TCA GTC CCT ATC       CAA    336                                                                        Asn Ala Asp Gly Asn Val Asp Tyr His M - #et Asn Ser Val Pro Ile Gln                      100    - #             105    - #             110                - -   CAA GAG ATT CTA GTG GTG CGC AAA GGG C - #AT CAA CCC TGC CCT AAT       TCA    384                                                                        Gln Glu Ile Leu Val Val Arg Lys Gly H - #is Gln Pro Cys Pro Asn Ser                  115        - #         120        - #         125                    - -   TTT AGG CTA GAG AAG ATG CTA GTG ACT G - #TA GGC TGC ACA TGC GTT       ACT    432                                                                        Phe Arg Leu Glu Lys Met Leu Val Thr V - #al Gly Cys Thr Cys Val Thr              130            - #     135            - #     140                        - -   CCC ATT GTT CAC AAT GTA GAC TAAAAG   - #                  - #                459                                                                       Pro Ile Val His Asn Val Asp                                                   145                - # 150                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 151 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - -   Met Thr Phe Arg Met Thr Ser Leu Val L - #eu Leu Leu Leu Leu Ser       Ile                                                                                 1              - # 5                 - # 10                 - # 15        - -   Asp Cys Ile Val Lys Ser Glu Ile Thr S - #er Ala Gln Thr Pro Arg       Cys                                                                                            20   - #               25   - #               30               - -   Leu Ala Ala Asn Asn Ser Phe Pro Arg S - #er Val Met Val Thr Leu       Ser                                                                                        35       - #           40       - #           45                    - -   Ile Arg Asn Trp Asn Thr Ser Ser Lys A - #rg Ala Ser Asp Tyr Tyr      Asn                                                                                    50           - #       55           - #       60                        - -   Arg Ser Thr Ser Pro Trp Thr Leu His A - #rg Asn Glu Asp Gln Asp      Arg                                                                                65               - #   70               - #   75               - #       80                                                                              - -   Tyr Pro Ser Val Ile Trp Glu Ala Lys C - #ys Arg Tyr Leu Gly Cys       Val                                                                                               - # 85                 - # 90                 - # 95        - -   Asn Ala Asp Gly Asn Val Asp Tyr His M - #et Asn Ser Val Pro Ile       Gln                                                                                           100    - #             105    - #             110               - -   Gln Glu Ile Leu Val Val Arg Lys Gly H - #is Gln Pro Cys Pro Asn       Ser                                                                                       115        - #         120        - #         125                    - -   Phe Arg Leu Glu Lys Met Leu Val Thr V - #al Gly Cys Thr Cys Val      Thr                                                                                   130            - #     135            - #     140                        - -   Pro Ile Val His Asn Val Asp                                              145                - # 150                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - -   CACAGGCATA CACAGGAAGA TACATTCA       - #                  - #                 28                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - -   TCTTGCTGGA TGGGAACGGA ATTCA        - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - -   ATACATTCAC AGAAGAGCTT CCTGCACA       - #                  - #                 28                                                                    __________________________________________________________________________

What is claimed is:
 1. An isolated human CTLA-8 protein comprising anamino acid sequence selected from the group consisting of:(a) the aminoacid of SEQ ID NO:2; (b) the amino acid sequence of SEQ ID NO:2 fromamino acids 11 to 163; (c) the amino acid sequence of SEQ ID NO:2 fromamino acids 29 to 163; (d) the amino acid sequence of SEQ ID NO:2 fromamino acids 31 to 163; (e) a fragment of SEQ ID NO:2, the fragmentcomprising amino acids 11-163 of SEQ ID NO:2; (f) a fragment of SEQ IDNO:2, and the fragment comprising amino acids 29-163 of SEQ ID NO:2; (g)a fragment of SEQ ID NO:2, the fragment comprising amino acids 31-163 ofSEQ ID NO:2.
 2. The protein of claim 1 comprising the amino acidsequence of SEQ ID NO:2.
 3. The protein of claim 1 comprising thesequence from amino acid 29 to 163 of SEQ ID NO:2.
 4. The protein ofclaim 1 comprising the sequence from amino acid 11 to 163 of SEQ IDNO:2.
 5. The protein of claim 1 comprising a fragment of SEQ ID NO:2,the fragment comprising amino acids 11-163 of SEQ ID NO:2.
 6. Theprotein of claim 1 comprising a fragment of SEQ ID NO:2, the fragmentcomprising amino acids 29-163 of SEQ ID NO:2.
 7. The protein of claim 1comprising a fragment of SEQ ID NO:2, the fragment comprising aminoacids 31-163 of SEQ ID NO:2.
 8. A human CTLA-8 protein producedaccording to a process which comprises the steps of:(a) growing aculture of a cell in a suitable culture medium, wherein the cell istransformed with a polynucleotide operably linked to an expressioncontrol sequence, wherein the polynucleotide is selected from the groupconsisting of:(aa) a polynucleotide comprising the nucleotide sequenceof SEQ ID NO:1 from nucleotide 55 to nucleotide 544; (ab) apolynucleotide comprising the nucleotide sequence of SEQ ID NO:1 fromnucleotide 146 to nucleotide 544; (ac) a polynucleotide comprising thenucleotide sequence of the full-length protein coding sequence of cloneB18 deposited under accession number ATCC 69868; (ad) a polynucleotideencoding the fill length protein encoded by the cDNA insert of clone B18deposited under accession number ATCC 69868; and (ae) a polynucleotidecomprising a nucleotide sequence varying from the sequence of thenucleotide sequence specified in (aa)-(ad) as a result of degeneracy ofthe genetic code; and (b) purifying the human CTLA-8 protein encoded bysaid polynucleotide from the culture.
 9. The human CTLA-8 protein ofclaim 8, wherein said polynucleotide comprises the nucleotide sequenceof SEQ ID NO:1 from nucleotide 55 to nucleotide
 544. 10. The humanCTLA-8 protein of claim 8 wherein said polynucleotide comprises thenucleotide sequence of SEQ ID NO:1 from nucleotide 146 to nucleotide544.
 11. The human CTLA-8 protein of claim 8, wherein saidpolynucleotide comprises the nucleotide sequence of the full-lengthprotein coding sequence of clone B18 deposited under accession numberATCC
 69868. 12. The human CTLA-8 protein of claim 8, wherein saidpolynucleotide comprises a polynucleotide encoding the full lengthprotein encoded by the cDNA insert of clone B18 deposited underaccession number ATCC
 69868. 13. A pharmaceutical composition comprisinga human CTLA-8 protein of claim 1 and a pharmaceutically acceptablecarrier.